Apoptosis is a physiological form of cell death that is required during normal development and plays a key role in controlling disease by mediating the elimination of cancerous or virus-infected cells. Many animal viruses have been found to regulate apoptosis (reviewed in Teodoro and Branton, J. Virol., 71:1739-1746 (1997); Roulston et al., Annu. Rev. Microbiol., 53: 577-628 (1999)). Inhibition of apoptosis can maximize viral replication efficiency and help evade an immune response. Conversely, induction of apoptosis near the end of virus replication can facilitate viral egress.
The development of novel and effective cancer therapies depends in part upon the discovery of agents that selectively destroy tumor cells while leaving normal cells intact. Several viruses have such selective intrinsic oncolytic activity or have been engineered to become oncolytic (reviewed in Kim et al., Nat. Med., 7:781-787 (2001)). For example, the Chicken Anemia Virus protein Apoptin can induce apoptosis in a variety of human malignant cell lines (Zhuang et al., Cancer Res., 55:486-489 (1995)). Two properties of Apoptin-induced cell death are important to note. First, Apoptin does not induce apoptosis in normal (untransformed) cells (reviewed in Pietersen and Noteborn, Adv. Exp. Med. Biol., 465:153-161 (2000)); and second, Apoptin-induced cell death is not dependent upon the p53 tumor suppressor (Danen-Van Oorschot et al., Proc. Natl. Acad. Sci., 94:5843-5847 (1997); Danen-Van Oorschot et al., Adv. Exp. Med. Biol., 457:245-249 (1999).). Thus, Apoptin represents a potential agent for the treatment of tumors that have lost their p53 status and are therefore refractory to many cancer therapies. Apoptin has shown efficacy in treating human xenografted tumors in mice and is currently being evaluated as a gene therapy agent to selectively destroy cancer cells (van der Eb et al., Cancer Gene Ther., 9:53-61 (2002)).
Apoptin contains no obvious functional motifs and has no homology with other proteins that would help elucidate its function. It does contain a canonical nuclear exportation signal (NES), and the subcellular location of Apoptin appears to be important for its activity: in normal cells, Apoptin is found predominantly in the cytoplasm, whereas in transformed and malignant cells it is located in the nucleus. The mechanism by which Apoptin differentially localizes to the nucleus in normal versus transformed cells is unknown, and whether this re-localization is critical for its apoptotic activity has not been determined. In addition, the molecular pathway by which Apoptin induces apoptosis has not been characterized.